Compositions and methods for healthy pregnancy

ABSTRACT

Compositions, kits and methods for the prevention of, for example, spontaneous abortion, preeclampsia, preterm labor or implantation failure during assisted reproduction are provided. The compositions, kits and methods provide an effective amount of granulocyte colony stimulating factor to prevent, for example, spontaneous abortion, preeclampsia, preterm labor or implantation failure of an embryo.

This application is a divisional application of U.S. patent applicationSer. No. 11/411,361, filed Apr. 24, 2006 which is a continuationapplication of PCT/US04/35468, filed Oct. 25, 2004, which claimspriority from U.S. Provisional Application Ser. No. 60/514,472, filedOct. 24, 2003. The entirety of all of the aforementioned applications isincorporated herein by reference.

FIELD

The present invention relates to methods of preventing spontaneousabortion or implantation failure during assisted reproduction andmethods of treating or preventing preeclampsia and preterm labor, andother forms of reproductive failure that present an immune systemaberration.

BACKGROUND

Conception, pregnancy and delivery require an intricate and delicateinterplay of physiology and anatomy. Implantation and placentation ofthe embryo are complex processes involving hormonal and anatomicalchanges in the mother and migration and cellular division of the embryo.

Spontaneous abortion occurs in 15% of diagnosed pregnancies in womenbetween fifteen and forty-five years of age. Recurrent spontaneousabortions are defined as the loss of three or more consecutivepregnancies and occur in about 3-4% of these women. The risk ofpregnancy loss increases from 15-20% in the first pregnancy to 40% afterone spontaneous abortion.

Although the majority of pregnancies lost in the first trimester are dueto fetal causes; spontaneous abortion, the loss of the product ofconception prior to the 20^(th) week of pregnancy, is a disorder ofunknown etiology. It has been theorized that spontaneous abortions are anatural rejection of a fetus with abnormalities incompatible with life,however, this theory has yet to be substantiated.

Risk factors for abortion include age, weight and overall health of thewoman. The prevalence of spontaneous abortion increases with increasingmaternal age, although not with gravidity. The risk begins to increaserapidly at age 35 years. The risk of spontaneous abortion at age 40 isapproximately twice that at age 20. As families are planned later andlater in life, the frequency of spontaneous abortion will only increasewithout effective methods of prevention.

Accompanying the rising age of hopeful parents is the increasing use ofassisted reproductive techniques such as in vitro fertilization, gameteintrafallopian tube transfer (GIFT), and the like. These techniques havetheir own attending risks, especially to the woman during ovarianhyperstimulation. Moreover, assisted reproduction and in vitrofertilization are costly, time consuming and have a high failure rate,resulting in pregnancy in only about 25% of cases. (see Merck Manual17th edition, 1999, Merck Research Laboratories, Whitehouse Station,N.J., p. 1995).

Threatened abortion generally presents as cramping and bleeding forwhich treatment is bed rest. This conservative treatment providespalliative care for the mother but does little to alter the outcome. Theuse of hormones is generally contraindicated due to the risk ofcongenital anomalies, including malformation of the vessels of the heartof the embryo and possible genital abnormalities in female offspring.

Preeclampsia and other hypertensive disorders of pregnancy are a leadingglobal cause of maternal and infant illness and death. Symptoms ofpreeclampsia include hypertension, edema and proteinuria with suddenweight gain, headaches and changes in vision. Preeclampsia can preventthe placenta from getting enough blood which can cause low birth weightand other problems for the baby. Although most women with preeclampsiastill deliver healthy babies, some develop eclampsia, a seriouscondition that threatens the life of the mother and the fetus.

The risk of preeclampsia is higher in women carrying multiple babies, inteenage mothers and in women older than age 40. Typically, preeclampsiaoccurs in the late 2nd or 3rd trimesters (middle to late pregnancy)though occasionally it occurs earlier. Preeclampsia affects about 5% ofall pregnancies.

Mild preeclampsia is conservatively treated with strict bed rest andvigilant monitoring of blood pressure. Progression of the disorder istreated with fluids, antihypertensives and magnesium sulfate butdelivery of the fetus provides the only remedy.

In addition to the physical toll of these disorders, the loss of adesired pregnancy takes a tremendous emotional toll on hopeful andexpectant parents. Loss of a pregnancy can lead to feelings ofinadequacy, hopelessness and guilt which can have a devastating effecton individuals and on a marriage.

New methods and compositions are always needed to reduce risksassociated with pregnancy to the health of the mother and fetus.Effective prevention of spontaneous abortion can allow women, especiallywomen at risk, to have successful pregnancies, and effective treatmentor prevention of preeclampsia can reduce or eliminate one of the commonhealth risks of pregnancy. Prevention of implantation failure duringassisted reproduction, can allow successful pregnancies, reduce therisks to the woman and save time and money. Prevention of preterm laborwill enhance the likelihood of normal births.

SUMMARY

To reduce the frequency of recurrent spontaneous abortion and thefailure of implantation during assisted reproduction, the presentinvention provides methods, compositions and kits comprising agranulocyte colony stimulating factor (GCSF or G-CSF) in an amounteffective to prevent spontaneous abortion or failed implantation of atransferred embryo. The present invention is based, in part, on thediscovery that GCSF can reduce or eliminate immune system mediatedaberrations that are associated with spontaneous abortion orimplantation failure. Based on this discovery, the present inventionalso provides methods of using GCSF mobilized peripheral blood stemcells to prevent spontaneous abortion or implantation failure, and thepresent invention also provides methods of using GCSF to treat orprevent preeclampsia or preterm labor.

In one aspect, the present invention provides methods of preventingspontaneous abortion. The methods can be administered to any femalesubject at risk for spontaneous abortion. Subjects at risk can beidentified according to the methods described herein or according tomethods known to practitioners in the art. Typically, the subject is inthe first or second trimester of pregnancy. In certain embodiments, thesubject is in the first 20 weeks of pregnancy. In certain embodiments,the subject is in the first or second months of pregnancy.

To prevent or reduce the risk of spontaneous abortion, aprophylactically effective amount of GCSF is administered to thesubject. The GCSF can be administered by any mode of administrationknown to those of skill in the art. In certain embodiments, the GCSF isadministered subcutaneously. The dose of GCSF can be determined by apractitioner of skill in the art as described in detail below. Incertain embodiments, a dose of 10 mcg (micrograms)/kg (body weight) upthrough 100-500 mg is administered daily to the subject. The dose can becontinued as long as necessary to prevent spontaneous abortion accordingto the judgment of the practitioner of skill in the art. In certainembodiments, the dose is administered through the first trimester ofpregnancy. In other embodiments, the dose is administered for four,three, two or one week. In particular embodiments, the dose isadministered for five, four, three, two or one day.

In another aspect, the present invention provides a method forpreventing spontaneous abortion by administering to a subject mobilizedperipheral blood stem cells. In this aspect, GCSF is administered to afirst female subject. Preferably, the subject is not pregnant. The GCSFcan optionally be administered along with chemotherapeutic orimmunosuppressive therapy. Following administration of GCSF,granulocytes are collected from the patient. These granulocytes areoptionally stored, typically by cryopreservation, for lateradministration. Preferably, the granulocytes comprise peripheral bloodstem cells, and in particular, CD34+ peripheral blood stem cells.

In this aspect of the invention, the stem cells, which can behistocompatible, are administered to a pregnant subject. In particularlypreferred embodiments, the administration is autologous, that is, therecipient subject is the same as the first female donor subject fromwhom the blood mononuclear cells were collected. However, theadministration can also be allogeneic, that is, the recipient subject isnot the same as the first female donor subject. An amount of the bloodmononuclear cells effective to prevent spontaneous abortion isadministered to the subject according to methods known to thepractitioner of skill in the art.

In another aspect, the present invention provides methods of preventingimplantation failure during assisted reproduction. The methods can beadministered to any female subject at risk for implantation failure.Subjects at risk can be identified according to the methods describedherein or according to methods known to the practitioner of skill in theart. In certain embodiments, the assisted reproduction is in vitrofertilization and transfer of an embryo.

In order to prevent implantation failure, a prophylactically effectiveamount of GCSF is administered to the subject. The GCSF can beadministered by any mode of administration known to those of skill inthe art. In certain embodiments, the GCSF is administeredsubcutaneously. The dose of GCSF can be determined by a practitioner ofskill in the art as described in detail below. In certain embodiments, adose of 10 mcg/kg up through 500 mg is administered daily to thesubject. The dose can be continued as long as necessary to preventimplantation failure according to the judgment of the practitioner ofskill in the art. In certain embodiments, the dose is administeredthrough the first trimester of pregnancy. In other embodiments, the doseis administered for four, three, two or one week. In particularembodiments, the dose is administered for five, four, three, two or oneday.

In another aspect, the present invention provides methods of preventingimplantation failure during assisted reproduction by administering to asubject GCSF mobilized peripheral blood stem cells, which can behistocompatible, as described above and in detail below. In this aspectof the invention, the blood stem cells are administered to a womanundergoing assisted reproduction. In particularly preferred embodiments,the administration is autologous, that is, the recipient subject is thesame as the first female donor subject from whom the blood cells werecollected. However, the administration can also be allogeneic, that is,the recipient subject is not the same as the first female donor subject.An amount of the blood stem cells effective to prevent implantationfailure during assisted reproduction is administered to the subjectaccording to methods known to practitioner of skill in the art.

In another embodiment, GCSF is administered to a subject undergoingassisted reproduction prior to or during ovarian stimulation to enhancethe quality, viability and/or number of oocytes available for retrieval.

In a further aspect, the present invention provides methods of treatingor preventing preeclampsia and preterm labor by administering to asubject in need thereof an effective amount of GCSF. The methods can beadministered to any female subject at risk for preeclampsia or pretermlabor. Subjects at risk can be identified according to the methodsdescribed herein or according to methods known to practitioners in theart. Typically, the subject is in the second or third trimester ofpregnancy.

To treat or prevent preeclampsia and preterm labor, an effective amountof GCSF is administered to the subject. The GCSF can be administered byany mode of administration known to those of skill in the art. Incertain embodiments, the GCSF is administered subcutaneously. The GCSFcan be formulated in any preparation known to those of skill in the artfor the mode of administration. The dose of GCSF can be determined by apractitioner of skill in the art as described in detail below. Incertain embodiments, a dose of 10 μg/kg up through 500 mg isadministered daily to the subject. The dose can be continued as long asnecessary to treat or prevent preeclampsia pr preterm labor according tothe judgment of the practitioner of skill in the art. In certainembodiments, the dose is administered through the end of pregnancy. Inother embodiments, the dose is administered for four, three, two or oneweek. In particular embodiments, the dose is administered for five,four, three, two or one day.

As described above and in detail in the sections below, the methods,kits and compositions of the invention have utility for preventingspontaneous abortion, implantation failure and for treating orpreventing preeclampsia or preterm labor.

DETAILED DESCRIPTION

As used herein, the following terms shall have the following meanings:

The terms “treat”, “treating” or “treatment” as used herein, refer to amethod of alleviating or abrogating a disorder and/or its attendantsymptoms. The terms “prevent”, “preventing” or “prevention”, as usedherein, refer to a method of barring a subject from acquiring a disorderand/or its attendant symptoms. In certain embodiments, the terms“prevent”, “preventing” or “prevention” refer to a method of reducingthe risk of acquiring a disorder and/or its attendant symptoms.

The term “spontaneous abortion” refers to delivery or loss of theproduct of conception before the 20th week of pregnancy. The termspontaneous abortion includes but is not limited to miscarriage,threatened abortion, inevitable spontaneous abortion, incompletespontaneous abortion, habitual or recurrent spontaneous abortion ormissed abortion.

The term “miscarriage” is synonymous with spontaneous abortion.

The term “threatened spontaneous abortion” refers to any bleeding orcramping of the uterus in the first 20 weeks of pregnancy.

The term “inevitable spontaneous abortion” refers to bleeding or ruptureof the membranes accompanied by pain and dilation of the cervix.

The term “incomplete spontaneous abortion” refers to expulsion of partof the products of conception or rupture of the membranes.

The term “habitual spontaneous abortion” or “recurrent spontaneousabortion” refers to three or more consecutive spontaneous abortions.

The term “missed abortion” refers to prolonged delay in expulsion of adead fetus.

The term “assisted reproduction” refers to clinical and laboratorytechniques used to enhance fertility in humans and animals, including,but not limited to, in vitro fertilization, GIFT, artificialinsemination and the like.

The term “in vitro fertilization” refers to the procedure involvingovarian hyperstimulation, oocyte retrieval from the mother-to-be or adonor, fertilization outside the subject's body, embryo culture andembryo transfer. As used herein, embryo transfer refers to the procedureinvolving transfer to a subject's uterus, of the developing or cleavingembryos or pre-embryos, also termed preimplantation embryos.

The term “implantation failure” refers to the failure of an embryoproduced by assisted reproduction to implant in the uterus of arecipient subject.

The term “preeclampsia” refers the development of hypertension withalburninuria or edema between the 20th week of pregnancy and the end ofthe first week postpartum. Any pregnant subject who develops a bloodpressure of 140/90 mm Hg, edema of the face or hands or albuminuria of≧1+ or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hgdiastolic (even if less than 140/90 mm Hg) is considered preeclampsic.

The term “colony stimulating factor” or “CSF” relates to a growth factorthat promotes and contributes to the maturity of cells, such as,hematopoietic and blood cells. Examples of CSF molecules include, butare not limited to, erythropoietin, GCSF, GMCSF, macrophage CSF,interleukin (IL)-3, IL-6 and stem cell factor.

The term “granulocyte-colony stimulating factor” or “G-CSF” refers tocompounds or factors that stimulate proliferation, differentiation,commitment and end cell functional activation of granulocytes in ananimal, including a human subject. G-CSF includes derivatives, mimetics,variants and chemically modified compounds or hybrids thereof asdescribed in U.S. Pat. Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183and 6,261,550, the contents of which are incorporated by reference inentireties. G-CSF is commercially available under the names filgrastim,(Neupogen®, Amgen and Granocyte®), Merck), pegfilgrastim (Neulasta®,Amgen) and lenograstim (Neutrogrin®, Chugai).

The term “granulocyte” refers to a blood cell containing granules,especially a leukocyte (white blood cell or corpuscle) containingneutrophil, basophil or eosinophil granules in its cytoplasm.

The term “granulocyte/macrophage colony stimulating factor” or “GMCSF”refers to compounds or factors that stimulate proliferation,differentiation, commitment and end cell functional activation ofmonocytes and granulocytes in an animal, including a human subject.GM-CSF includes derivatives, mimetics, variants and chemically modifiedcompounds or hybrids thereof as described in, for example, U.S. Pat.Nos. 5,895,646; 5,891,429 and 5,908,763; the contents of which areincorporated by reference in entireties. GM-CSF is commerciallyavailable under the trade names Leukine®, Berlex and Leucomax®, Wyeth.

The term “macrophage” relates to a mononuclear, phagocytic cell that canexit the circulation and enter tissue spaces.

The term “therapeutically effective amount” refers to that amount of anactive agent being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated.

The term “preterm labor” also known as premature labor, refers to thebeginning of regular contractions that cause the cervix to begindilation and effacement before the 37th week of pregnancy.

The term “prophylactically effective amount” refers to that amount of anactive agent being administered sufficient to prevent the disorder orprevent one or more symptoms of the disorder being treated. In certain,embodiments, the term “prophylactically effective amount” refers to thatamount of an active agent being administered sufficient to reduce therisk of the disorder or one or more symptoms of the disorder.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (such as humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In preferred embodiments, thesubject is a human female.

The term “label” refers to a display of written, printed or graphicmatter on the immediate container of an article, for example the writtenmaterial displayed on a vial containing a pharmaceutically active agent.

The term “labeling” refers to all labels and other written, printed orgraphic matter on any article or any of its containers or wrappers oraccompanying such article, for example, a package insert orinstructional videotapes or computer data storage devices, such as CDsand DVDs, accompanying or associated with a container of apharmaceutically active agent.

The present invention is directed to methods of preventing spontaneousabortion and implantation failure and methods of treating or preventingpreeclampsia described in detail below.

In one embodiment, the present invention provides methods of preventingspontaneous abortion by administering to a subject in need thereof aprophylactically effective amount of a GCSF.

While not intending to be bound by any particular theory of operation,it is believed that spontaneous abortion and recurrent spontaneousabortion are caused or associated with inappropriate immune responses ina pregnant subject. In particular, it is believed that subjects at riskfor spontaneous abortion and recurrent spontaneous abortion presentinappropriate immune cytokines associated with a T-helper 1 (Th1) immuneresponse known to those of skill in the art. (See, Kwak-Kim et at, 2003,Hum. Reprod. 18(4): 676-773.) In contrast, subjects that have healthypregnancies typically present immune cytokines associated with aT-helper 2 (Th2) immune response. It is believed that administration ofGCSF can reduce the inappropriate Th1 response and/or increase aT-helper 2 (Th2) immune response in a subject. This invention is thusbased, in part, on the discovery that administration of GCSF can shift asubject's immune response towards a healthy Th2 response duringpregnancy and thereby reduce or eliminate the risk of spontaneousabortion.

The subject can be any mammalian subject at risk for a spontaneousabortion. In particularly preferred embodiments, the subject is a humanfemale. In certain embodiments, the subject has previously had one ormore spontaneous abortions. In further embodiments, the subject haspreviously had two or more spontaneous abortions. In other embodiments,the subject has had recurrent spontaneous abortions, i.e., three or morespontaneous abortions.

In further embodiments, the subject can be any subject in a populationat risk for spontaneous abortion. For instance, the subject can be ahuman female in an age group at risk for spontaneous abortion. Inparticular embodiments, the subject can be a human female greater than35 years of age, greater than 40 years of age or greater than 45 yearsof age. In other particular embodiments, the subject can be a humanfemale less than 20 years of age or less than 15 years of age. However,essentially a woman of any age that presents with a reproductiveinfirmity, such as spontaneous abortion, preeclampsia and preterm labor,is a candidate for obtaining the materials and methods of the instantinvention.

In further embodiments, the subject can also be in any other populationat risk for spontaneous abortion as determined by a practitioner ofskill in the art. In certain embodiments, the subject is threateningabortion. In other embodiments, the subject is obese, morbidly obese,has overall poor health or comorbid conditions that indicate a risk ofspontaneous abortion to the skilled practitioner. In certainembodiments, these conditions can be incompetent cervix, uterineanomalies, hypothyroidism, diabetes mellitus, chronic nephritis, acuteinfection, use of illicit drugs (such as cocaine or crack), immunologicproblems, severe emotional shock and viral infection (especiallycytomegalovirus, herpes virus and rubella) (see Merck Manual 17thedition, 1999, Merck Research Laboratories, Whitehouse Station, N.J., p.2053). In certain embodiments, the subject has had an implantationfailure during a previous assisted reproduction procedure. Othersubjects at risk include those with unusually high Th1 immune responsesor unusually low Th2 immune responses. In further embodiments, thesubject can also be in any other population at risk for spontaneousabortion as determined by a practitioner of skill in the art.

In certain embodiments, the GCSF is administered to the subject prior topregnancy. For instance, the GCSF is administered to a subject that isplanning or attempting to become pregnant. In other embodiments, theGCSF is administered to a pregnant subject. The GCSF can be administeredat any time during the first or second trimester of pregnancy. Inpreferred embodiments, the GCSF is administered during the first 20weeks of pregnancy.

The GCSF can be any GCSF known to those of skill in the art to beeffective in modulating the immune system of the subject. Thus, a rangeof modifications can be made to the wild-type GCSF molecules so long asthe known immune system modulating activity of the GCSF is maintained.There are a number of assays that can be used to ensure that any onemodified GCSF retains the desired immune system modulating activity. TheGCSF can be formulated according to any formulation for administrationknown to those of skill in the art. Plural types of GCSF molecules canbe administered in the practice of the instant invention. The pluralGCSF molecules can be administered consecutively or sequentially. Inpreferred embodiments, the GCSF formulation is the commerciallyavailable filgrastim (Neupogen® Amgen), pegfilgrastim (Neulasta®, Amgen)or lenograstim (Neutrogrin®g, Chugai). Other effective GCSF moleculesand formulations are described in detail in the sections below.

The GCSF formulation is administered in a prophylactically effectiveamount, i.e., an amount effective to reduce or eliminate the risk ofspontaneous abortion in the subject. The amount can be determined by theskilled practitioner guided by the description herein and the knowledgein the art. In preferred embodiments, the amount can be any amount ofGCSF that reduces the Th1 response of the subject. In furtherembodiments, the amount can be any amount sufficient to increase orinitiate a Th2 response in the subject. Assays to determine Th1 and Th2responses in the subject are well known to those of skill in the art(See Schust and Hill, 1996, J. Soc. Gynecol Investig. 3:259-61, Xing etal., 2001, Chin. Med. J. 114:921-4, Raghupathy et al., 1999, CellImmunol. 196:122-30, Mauri et al., 1996, J. Immunol. 26:1511-8, Doncorliet al., 1997, Eur. J. 1 mm. 27:1451-8, Raziuddin, 1998, J. Rheumatol25:329-33, Moverare et al., 2000, Allergy 55:171-5). In particularembodiments, a dose of 1 to 100 mcg/kg, 1 to 20 mcg/kg or about 10mcg/kg is administered to the subject. In another embodiment, at least25 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 125 mg,at least 150 mg, at least 175 mg, at least 200 mg, at least 300 mg ormore is administered daily.

The dose can be administered to the subject daily until the risk ofspontaneous abortion is reduced or eliminated and as long as no symptomsof toxicity are presented. In certain embodiments, the dose isadministered daily through the second trimester of pregnancy. In furtherembodiments, the dose is administered daily through the 20th week ofpregnancy. In particular embodiments, the dose is administered daily forfour, three, two or one week during the first or second trimester ofpregnancy. In particular embodiments, the dose is administered for fiveconsecutive days during the first or second trimester of pregnancy. Forexample, the five consecutive days can be in the first or second week ofpregnancy.

The GCSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration. Other effective modes ofadministration are described in detail in the sections below.

In another aspect, the present invention provides methods of preventingspontaneous abortion by administering to a subject in need thereof aneffective amount of colony stimulating factor-mobilized peripheral bloodstem cells.

While not intending to be bound by any particular theory of operation,as discussed above, it is believed that spontaneous abortion is causedor associated with an inappropriate Th1 immune response. It is believedthat administration of peripheral blood stem cells can preventspontaneous abortion by reducing the inappropriate Th1 immune responseand/or increasing a Th2 immune response in a subject at risk forspontaneous abortion. It has been observed that GCSF can mobilizeperipheral blood stem cells, and that these stem cells, whenadministered to a subject, can shift the subject's immune responsetoward a Th2 response. Thus, the present invention provides methodsbased, in part, on the discovery that GCSF mobilized stem cells canprevent spontaneous abortion.

The subject can be any mammalian subject at risk for a spontaneousabortion as described in the section above. In particularly preferredembodiments, the subject is a human female. In certain embodiments, thesubject has previously had one or more, two or more, or three or morespontaneous abortions. In certain embodiments, the subject isthreatening abortion or has had more than one threatened abortion. Infurther embodiments, the subject can be a human female greater than 35years of age, greater man 40 years of age or greater than 45 years ofage. In other particular embodiments, the subject can be a human femaleless than 20 years of age or less than 15 years of age. However, anywoman of any age with imperfect reproductive fitness arising from, forexample, spontaneous abortion, preeclampsia or preterm labor can obtainthe benefits of the instant invention. In other embodiments, the subjectis obese, morbidly obese, has overall poor health or comorbid conditionsthat indicate a risk of spontaneous abortion to the skilledpractitioner. In certain embodiments, these conditions can beincompetent cervix, uterine anomalies, hypothyroidism, diabetesmellitus, chronic nephritis, acute infection, use of illicit drugs (suchas cocaine or crack), immunologic problems, severe emotional shock andviral infection (especially cytomegalovirus, herpes virus and rubella)(see Merck Manual 17th edition, Merck Research Laboratories, p. 2053).Other subjects at risk include those with unusually high Th1 immuneresponses or unusually low Th2 responses. In further embodiments, thesubject can also be in any other population at risk for spontaneousabortion as determined by a practitioner of skill in the art.

In the methods of this aspect of the invention, GCSF is administered toa first subject to mobilize peripheral blood stem cells that are to becollected for administration to a subject at risk for spontaneousabortion. In preferred embodiments, the peripheral blood stem cells,which can be histocompatible, comprise CD34+ peripheral blood stemcells. Preferably, the first subject is a female that is not pregnant.In preferred embodiments, the methods are autologous. In theseembodiments, peripheral blood stem cells are collected from the firstsubject and then administered to the same first subject. For instance,the cells can be collected while the subject is not pregnant, stored andlater administered to the same subject before or after a positivepregnancy test. In other embodiments, the methods are allogeneic. Theperipheral blood stem cells are collected from a first subject andadministered to a second subject.

The GCSF can be any GCSF known to one of skill in the art to beeffective in mobilizing peripheral blood stem cells. The G-CSF can beformulated according to any formulation for administration known tothose of skill in the art. In preferred embodiments, the G-CSFformulation is the commercially available (Neupogen®® Amgen),pegfilgrastim (Neulasta® Amgen) or lenograstim (Neutrogrin®, Chugai).Other effective G-CSF molecules and formulations are described in detailin the sections below.

The G-CSF formulation is administered in an amount effective to mobilizeperipheral blood stem cells for collecting from the subject. The amountcan be determined by the skilled practitioner guided by the descriptionherein and the knowledge in the art. In preferred embodiments, theamount can be any amount of G-CSF that mobilizes peripheral blood stemcells. In particular embodiments, a dose of 1 to 100 mcg(micrograms)/kg, 1 to 20 mcg/kg or about 10 μg/kg is administered to thesubject.

In other embodiments, at least 25 mg; at least 50 mg; at least 75 mg; atleast 100 mg; at least 125 mg; at least 150 mg; at least 175 mg; atleast 200 mg; at least 300 mg or more GCSF is administered daily.

The dose can be administered to the subject daily for any time periodnecessary to mobilize peripheral blood stem cells as known to those ofskill in the art. In certain embodiments, the dose is administered dailyfor four, three, two or one week. In particular embodiments, the dose isadministered for five consecutive days. In other embodiments, the doseis administered daily for five, four, three, two or one day.

The G-CSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration. Other effective modes ofadministration are described in detail in the sections below.

In certain embodiments, the G-CSF is administered as a monomerapy. Inother embodiments, the G-CSF is administered with at least one otheractive compound. The G-CSF and at least one other active compound can beadministered simultaneously or sequentially, continuously orintermittently. For example, the other active ingredient can beadministered according to the doses and schedules known to those ofskill in the art while the G-CSF is administered according to themethods described herein. The at least one other active compound can beanother CSF. The other active compound can be a drug currently used totreat the conditions of interest. The other active compound can be adrug that is an immunosuppressant. In preferred embodiments, the atleast one other active ingredient is a chemotherapeutic ornon-myeloablative immunosuppressive agent. For example, the other activeingredient can be cyclophosphamide or a purine nucleoside analog such ascladribine and fludararbine. Preferred chemotherapeutic ornommyeloablative immunosuppressive agents are described in detail in thesections below. The other active agent could also be another knownimmunosuppressive/antiinflammatory agent such as vitamin D (or one ofits analogs) or aspirin. In addition, the at least one other activeagent could be one that is currently widely used for the treatment ofTh1 cytokine excess in pregnancy, such as heparin, IVIG or progesterone.

After an effective dose of G-CSF has been administered to the firstsubject, granulocytes are collected from the first subject according toany method known to those of skill in the art. For example, whole bloodcan be collected from the first subject by any method known to those ofskill in the art. Granulocytes, including peripheral blood stem cells,can be isolated from the whole blood by any method known to those ofskill in the art such as cytophoresis or including, leukophoresis (alsoknown as cytapharesis and leukapharesis). (See, Guidelines forTherapeutic Hemapheresis, revised May 1993, American Association ofBlood Banks, Bethesda, Md.). Preferably, the granulocytes in thesemethods of the invention comprise peripheral blood stem cells, inparticular, CD34+ peripheral blood stem cells. Assays for CD34+ cellsare within the skill of those in the art (see e.g., Link and Arseniev1997, Leuk. Lymphoma 26:451-65, Vogel et al., 2000, Stem Cells 18:87-92,Dreger et al., 1994, Br. J. Haematol. 87:609-613; and Berenson et al.,1996, Cancer Invest. 14:589-96).

In certain embodiments, these granulocytes can be stored for lateradministration to the first subject or to another subject. Althoughcryopreservation is the primary method of storing granulocytes, othermethods are being developed for the long term storage of blood cells andcan be used in the methods of invention. (See, for example, U.S. Pat.No. 6,150,085, Papadimitriou et al., 2000, J. Clin. Apheresis15:236-24:236-241; Arpaci et al., 2000, Jpn. J. Clin. Oncol 30:154-88).Formulations and methods of storage such as cryogenic preservation arewell known to those of skill in the art. The collected granulocytes canoptionally be formulated for administration to a subject prior tostorage or after storage.

In these methods of the invention, the collected granulocytes areadministered to a subject to prevent spontaneous abortion. In certainembodiments, the cells are administered to the subject prior topregnancy. For instance, the cells can be administered to a subject thatis planning or attempting to become pregnant. In other embodiments, thecells are administered to a pregnant subject. The cells can beadministered at any time during the first or second trimester ofpregnancy. In preferred embodiments, the cells are administered duringthe first 20 weeks of pregnancy.

The cells are administered in a prophylactically effective amount, anamount effective to reduce or eliminate the risk of spontaneous abortionin the subject. The amount can be determined by the skilled practitionerguided by the description herein and the knowledge in the art. Inpreferred embodiments, the amount can be any amount of cells that reducethe Th1 response of the subject. In further embodiments, the amount canbe any amount sufficient to increase or initiate a Th2 response in thesubject Assays to determine Th1 and Th2 responses in the subject arewell known to those of skill in the art (See e.g., Schust and Hill,1996, J. Soc. Gynecol Investig. 3:259-61, Xing et al., 2001, Chin. Med.J. 114:921-4, Raghupathy et al., 1999, Cell Immunol 196:122-30, Mauri etal., 1996, Eur. J. Immunol. 26:1511-8, Doncarli et al., 1997, Eur. J.Immunol. 27:1451-8, Raziuddin, 1998, J. Rheumatol 25:329-33, Moverare etal., 2000, Allergy 55:171-5). In particular embodiments, about 250-750ml of collected leukapheresis material or about 5 to 15×106 CD34+ cellsis administered to the subject.

The cells can be administered to the patient daily until the risk ofspontaneous abortion is reduced or eliminated and as long as no symptomsof toxicity are presented. In certain embodiments, the cells areadministered daily through the second trimester of pregnancy. In furtherembodiments, the cells are administered daily through the 20th week ofpregnancy. In particular embodiments, the cells are administered dailyfor four, three, two or one week during the first or second trimester ofpregnancy.

The peripheral blood stem cells can be administered according to anymethod of administration known to those of skill in the art. Preferredmethods of administration of blood stem cells include intravenousadministration.

In another aspect, the present invention provides methods of preventingembryo implantation failure during assisted reproduction byadministration to a subject in need thereof a prophylactically effectiveamount of granulocyte colony stimulating factor.

In vitro fertilization is an assisted procedure to overcome fertilityproblems caused by, for example, tubal disease, endometriosis,oligospermia, sperm antibodies and unexplained infertility. Theprocedure can include ovarian hyperstimulation with ‘fertility drugs’such as ovarian stimulants like clomiphene citrate andgonadotropin-releasing hormones. Hyperstimulation of the ovaries caninduce growth of the egg (oocyte) and its encasing cells, collectivelyalso termed the ovarian follicles. After sufficient follicular growth,final follicular maturation is induced and oocytes are retrieved orharvested. The oocytes are fertilized in vitro with sperm and theembryos cultured. A small number of embryos, generally 2-4, are thentransferred to the uterus. Despite the transfer of multiple embryos, theterm pregnancy rate is only about 25%. (see Merck Manual 17th edition,1999, Merck Research Laboratories, Whitehouse Station, N.J., p. 1995).

While not intending to be bound by any particular theory of operation,it is believed that implantation failure during assisted reproduction iscaused or associated with inappropriate immune responses in the embryorecipient. In particular, it is believed that subjects at risk forembryo implantation failure present with an overproduction of T-helper 1(Th1) cytokines and underproduction of T-helper 2 (Th2) cytokines.Positive correlations in human and animal models have been demonstrated,(see, Kwak-Kim et al., 2003, Human Reproduction 18:767-73, Krishnan etal., 1996, J. Immunol. 156:653-62) but remain controversial (see,Chaouat et al., 2003, J. Reproductive Immunol. 59:205-17). The Th1cytokine associated with overproduction can be interferon-γ (INF-γ). TheTh2 cytokines associated with underproduction can be interleukins 10 and4 (IL-10 and IL-4).

In the methods of prevention, the G-CSF is typically administered untilimplantation of the embryo to the uterine wall is achieved, until therisk of failed implantation is reduced or eliminated or according to thejudgment of a practitioner of skill in the art.

In certain embodiments, the administration is continued until pregnancyis confirmed. In certain embodiments, the administration is startedabout the time of ovarian hyperstimulation and continued until about 3days, about 5 days, about 7 days, about 10 days, about 12 days, about 14days or about 30 days after embryo transfer to the subject's uterus. Incertain embodiments, the administration is started about the time ofovarian hyperstimulation and continued until about the end of the firsttrimester. In another embodiment, the dose is administered for fiveconsecutive days about the time of embryo transfer to the subject'suterus. In certain embodiments, the administration is continued untilthe subject presents a normal Th1 immune response for a pregnant subjector a normal Th2 immune response for a pregnant subject or both,according to the judgment of a practitioner of skill in the art.

In certain embodiments, a prophylactically effective amount of GCSF isadministered to a subject at risk of embryo implantation failure. Incertain embodiments, a subject at risk is a subject that has failed oneor more in vitro fertilization procedures. In further embodiments, thesubject can also be in any other population at risk for failed embryoimplantation as determined by a practitioner of skill in the art. Incertain embodiments, the subject has previously failed assistedreproduction. In another embodiment, the subject has had one or moreprevious spontaneous abortions. Other subjects at risk include thosewith unusually high Th1 immune responses or unusually low Th2 immuneresponses. In further embodiments, the subject can also be in any otherpopulation at risk for failed embryo implantation as determined by apractitioner of skill in the art.

In certain embodiments, the G-CSF is administered to the subject priorto embryo transfer. For instance, the G-CSF is administered to a subjectthat is planning or attempting to become pregnant via assistedreproduction. Thus, the GCSF can be administered to the mother-to-beduring the superovulation procedure or if ova are donated, prior toimplantation of the embryos. In other embodiments, the G-CSF isadministered to a subject after retrieving or harvesting oocytes. Inanother embodiment, the retrieved oocytes and the embryos are maintainedand cultured in medium containing GCSF prior to being instilled in themother-to-be. The G-CSF can be administered at any time during theassisted reproduction or in vitro fertilization process.

The G-CSF can be any G-CSF known to those of skill in the art to beeffective in modulating the immune system of the subject. The G-CSF canbe formulated according to any formulation for administration known tothose of skill in the art. In preferred embodiments, the G-CSFformulation is the commercially available filgrastim (Neupogen®, Amgen),pegfilgrastim (Neulasta®, Amgen) or lenograstim (Neutrogrin®, Chugai).Other effective G-CSF molecules and formulations are described in detailin the sections below.

The G-CSF formulation is administered in a prophylactically effectiveamount, i.e., an amount effective to reduce or eliminate the risk ofimplantation failure in the subject. The amount can be determined by theskilled practitioner guided by the description herein and the knowledgein the art. In preferred embodiments, the amount can be any amount ofG-CSF that reduces the Th1 response of the subject. In furtherembodiments, the amount can be any amount sufficient to increase orinitiate a Th2 response in the subject. In particular embodiments, adose of 1 to 100 mcg/kg, 1 to 20 mcg/kg or about 10 mcg/kg isadministered daily to the subject. In other embodiments, at least 25 mg;at least 50 mg; at least 75 mg; at least 100 mg; at least 125 mg; atleast 150 mg; at least 175 mg; at least 200 mg; at least 300 mg or moreGCSF is administered dairy.

The G-CSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration. Other effective modes ofadministration are described in detail in the sections below.

In another aspect, the present invention provides methods of preventingimplantation failure during assisted reproduction by administering to asubject G-CSF mobilized peripheral blood stem cells. In this aspect,G-CSF is administered to a first female subject and peripheral bloodstem cells, particularly CD34+ peripheral blood stem cells are collectedfrom the first subject as described herein. In particularly preferredembodiments, the administration is autologous. However, theadministration can also be allogeneic, i.e., the recipient subject isnot the same as the first female donor subject. An amount of the bloodstem cells, which can be histocompatible, effective to preventimplantation failure during assisted reproduction is administered to thesubject according to methods described above. In another aspect, thepresent invention provides methods of preventing implantation failureduring assisted reproduction by administering G-CSF mobilized peripheralblood stem cells to a subject prior to ovarian hyperstimulation. Inanother aspect, the present invention provides methods of administeringabout 250-750 ml of collected leukapharesis material or about 5 to15×106 CD34 cells to the subject. In another aspect, the presentinvention provides for administration of peripheral blood stem cellsfrom about the time of ovarian hyperstimulation and continued untilabout 3 days, about 5 days, about 7 days, about 10 days, about 12 days,about 14 days or about 30 days after embryo transfer to the subject'suterus.

In a further aspect, the present invention provides methods of treatingor preventing preeclampsia or preterm labor by administering to asubject in need thereof an effective amount of granulocyte colonystimulating factor.

While not intending to be bound by any particular theory of operation,it is believed that preeclampsia and preterm labor is caused orassociated with inappropriate immune responses in a pregnant subject. Inparticular, it is believed that subjects at risk for preeclampsia orpreterm labor present inappropriate immune cytokines associated with aT-helper 1 (Th1) immune response known to those of skill in the art. Incontrast, subjects that have healthy pregnancies typically presentimmune cytokines associated with a T-helper 2 immune response. It isbelieved that administration of G-CSF can reduce the inappropriate Th1response and/or increase a Th2 immune response in a subject. Thisinvention is thus based, in part, on the discovery that administrationof G-CSF can shift a subject's immune response towards a healthy Th2response during pregnancy and thereby treat or prevent preeclampsia orpreterm labor.

In the methods of treatment, G-CSF is administered to a subjectpresenting one or more symptoms of preeclampsia or preterm labor. Thesubject can be any subject that presents any of the symptoms ofpreeclampsia during pregnancy such as hypertension, swelling or edemaand excessive protein in the urine. For example, the subject can be anysubject that develops hypertension with albuminuria or edema between the20th week of pregnancy and the end of the 1st week postpartum.Particular subjects include pregnant females who develop a bloodpressure of 140/90 mm Hg, edema of the face or hands or albuminuria of≧1+ or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hgdiastolic (even if less than 140/190 mm Hg) between the 20th week ofpregnancy and the end of the 1st week postpartum. Particularly preferredsubjects are human females.

In the methods of treatment, the G-CSF is typically administered untilthe symptoms of preeclampsia or preterm labor are alleviated or reducedas long as the therapeutic benefit outweighs the risk of adverse eventsaccording to the judgment of a practitioner of skill in the art. Thedosing can continue as long as the subject displays no toxic effects ofthe administration according to the judgment of a practitioner of in theart. In certain embodiments, the treatment is continued until thesubject presents a normal Th1 immune response for a pregnant subject ora normal Th2 response for a pregnant subject, or both, according to thejudgment of a practitioner of skill in the art.

In the methods of prevention, G-CSF is administered to a subject at riskfor developing preeclampsia or preterm labor. The subject can be anymammalian subject at risk for preeclampsia or preterm labor. Subjects atrisk include subjects carrying multiple babies, subjects younger thanage 20 and subjects older than age 40. Further subjects include thosepregnant for the first time (primigravida), subjects with preexistinghypertension and subjects with preexisting vascular disease. Othersubjects at risk include those with unusually high Th1 immune responsesor unusually low Th2 immune responses. In particularly preferredembodiments, the subject is a human female.

In the methods of prevention, G-CSF is administered as long as thesubject is at risk for preeclampsia and as long as the therapeuticbenefit outweighs the risk of adverse events and also, so long as notoxicity is observed according to the judgment of a practitioner ofskill in the art. In certain embodiments, G-CSF is administered for theduration of the pregnancy. In particular embodiments, administration isprovided in the 2nd and 3rd trimester of pregnancy. In furtherembodiments, administration is continued after delivery for about one,about two, about three, about four, about five, about six, about sevenor about eight weeks post partum. In certain embodiments, the treatmentis continued until the subject presents a normal Th1 immune response fora pregnant subject or a normal Th2 immune response for a pregnantsubject, or both, according to the judgment of a practitioner of skillin the art.

The G-CSF can be any G-CSF known to those of skill in the art to beeffective in modulating the immune system of the subject as described inthe sections above. The GCSF can be formulated according to anyformulation for administration known to those of skill in the art. Inpreferred embodiments, the G-CSF is the commercially availablefilgrastim or pegfilgrastim. Other effective G-CSF molecules andformulations are described in detail in the sections below.

The G-CSF formulation is administered in a prophylactically ortherapeutically effective amount, i.e., an amount effective to preventor reduce or eliminate the preeclampsia or the symptoms of preeclampsiain the subject. The amount can be determined by the skilled practitionerguided by the description herein and the knowledge in the art. Inpreferred embodiments, the amount can be any amount of G-CSF thatreduces the Th1 response of the subject. In further embodiments, theamount can be any amount sufficient to increase or initiate a Th2response in the subject. Assays to determine Th1 and Th2 responses inthe subject are well known to those of skill in the art (See e.g.,Schust and Hill, 1996, J. Soc. Gynecol. Investig. 3:259-61, Xing et al.,2001, Chin. Med. J. 114:921-4, Raghupathy et al., 1999, Cell Immunol.196:122-30, Mauri et al., 1996 Eur. J. Immunol. 26:1511-8, Doncarli etal., 1997, Eur. J. Immunol. 27:1451-8, Raziuddin, 1998, J. Rheumatol.25:329-33, Moverare et al., 2000, Allergy 55:171-5). In particularembodiments, a daily dose of 1 to 100 mcg/kg, 1 to 20 mcg/kg or about 10mcg/kg is administered to the subject. In other embodiments, at least 25mg; at least 50 mg; at least 75 mg; at least 100 mg; at least 125 mg; atleast 150 mg; at least 175 mg; at least 200 mg; at least 300 mg or moreGCSF is administered daily.

The G-CSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration. Other effective modes ofadministration are described in detail in the sections below.

While generally a medical history will serve to ascertain candidatesubjects in need of treatment as described herein, diagnostic assays canbe used to ascertain subjects presenting with reproductiveinefficiencies that are correlated with particular immunologicparameters. As noted herein, patients with repeated spontaneous abortionor miscarriage, preeclampsia, preterm labor and the like present withparticular profiles of their immune system status. Thus, subjects withhigh Th1 cell number or cell activity and/or reduced Th2 cell number orcell activity, or an aberrant ratio of the two may be candidates forobtaining the instant treatment of interest.

Hence, a diagnostic assay of interest is one that determines whether Th1cell number or cell activity is enhanced. Another assay of interest isone that determines whether Th2 cell number or activity is decreased.Yet another assay of interest is one that determines a higher ratio ofTh1 cell number to Th2 cell number, or Th1 cell activity to Th2 cellactivity.

A number of known assays, for example, immunoassays or bioassays, can beused to make such determinations. For example, γ interferon, tumornecrosis factor β and IL-2 are markers of Th1 cells. Thus, assays forone or more of such cell-specific markers can provide the basis toconclude a higher than normal Th1 status. As to Th2, IL-4, IL-5, IL-6,IL-10 and IL-13 are known markers of that cell type. Thus, assays forone or more of such cell-specific markers can provide the basis toconclude a higher than normal Th2 status.

As described in detail above, the present invention provides methods ofadministering an effective amount of granulocyte colony stimulatingfactor (G-CSF) to prevent abortion, implantation failure during assistedreproduction or to treat or prevent preeclampsia or preterm labor.

The G-CSF administered in the methods of the invention can be any G-CSFknown to one of skill in the art without limitation. In certainembodiments, the G-CSF can be any G-CSF or any derivative, variant,mimetic, chemically modified version or hybrid thereof, as described inU.S. Pat. Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183 and 6,261,550,the contents of which are hereby incorporated by reference in theirentireties. In further embodiments, the G-CSF can be administered in theform of a nucleotide sequence encoding G-CSF or expression vectorsencoding G-CSF described in U.S. Pat. No. 5,422,248, the content ofwhich is hereby incorporated by reference in its entirety.

In certain embodiments, the G-CSF is a commercially available G-CSFavailable as a pharmaceutical composition, suitable for administrationto an animal, including a human. Such commercially availablepharmaceutical compositions can be, for example, filgrastim (Neupogen®,Amgen), pegfilgrastim (Neulasta®, Amgen)) or lenograstim (Neutrogrin®,Chugai).

Filgrastim and lenograstim are useful for promoting neutrophilproliferation and is generally administered to individuals in need toincreased neutrophils, for example, patients undergoing chemotherapy.Filgrastim and lenograstim are indicated for myelosuppressivechemotherapy, bone marrow transplant, peripheral blood progenitor cellcollection and severe chronic neutropenia. Off label uses includetreatment of neutropenia in AIDS patients, aplastic anemia, hairy cellleukemia, myelodysplasia, drug-induced and congenital agranulocytosisand alloimmune neonatalneutropenia.

The usual treatment of myelosuppressive is 5 mcg/kg/day, once dailyeither by bolus subcutaneously or short (15-30 minute) intravenousinfusion or by continuous subcutaneous or intravenous infusion.Administration is once daily starting no earlier man 24 hours afterchemotherapy and continues for 14 days or until the individual'sabsolute neutrophil count is 10,000/mm³. For patients undergoing bonemarrow transplant, the usual dose is 10 mcg/kg/day administered as anintravenous infusion over 4-24 hours or as a continuous 24 hoursubcutaneous infusion. The first dose is generally administered at least24 hours after chemotherapy and at least 24 hours after bone marrowinfusion. During recovery, the dose is adjusted by 5 mcg/kg/daytitrating to the patient's absolute neutrophil count. Filgrastim dosingfor peripheral blood progenitor cells generally begins at 10 μg/kg/daysubcutaneously either as a bolus or continuous infusion. It isrecommended that filgrastim be given for at least four days beforeleukapheresis and continued until the last leukapheresis procedure.Doses of filgrastim for congenital neutropenia are 6 mcg/kgsubcutaneously twice daily while idiopathic or cyclic neutropenia isgenerally treated with a dose of 5 mcg/kg subcutaneously once daily.

Pegfilgrastim is a monomethoxypolyethylene glycol conjugate offilgrastim. The pharmaceutical composition is commercially available aspreservative free solutions of 10 mg/ml pegfilgratim in prefilledsingle-dose syringes. Pegfilgrastim is indicated to decrease infectionsin patients with febrile neutropenia undergoing myelosuppressivechemotherapy. Recommended dosing is a single 6 mg subcutaneous injectionadministered once per chemotherapy cycle.

In certain embodiments described above, the present invention providesmethods administering to a subject in need thereof an effective amountof G-CSF as monotherapy. In other embodiments, the present inventionprovides methods of administering to a subject an effective amount ofG-CSF in combination with at least one other active agent. Other activeagents include myeloablative immunosuppressive therapy.

In one aspect, the non-myeloablative immunosuppressive therapy can beany non-myeloablative immunosuppressive therapy available to one ofskill in the art. In one aspect, the immunosuppressive therapy can beone or more antineoplastic or chemotherapeutic agents including, but notlimited to, alkylating agents, antimetabolites, antimitotic agents,epipodophyllotoxins, antibiotics, hormones, enzymes, platinumcoordination complexes, anthracenediones, substituted ureas,methylhydrazine derivatives and topoisomerases, as described, forexample, in U.S. Pat. No. 6,162,417, incorporated herein by reference inits entirety. Examples of such antineoplastic or chemotherapeutic agentsinclude, but not limited to, chlorambucil (Leukeran®, GlaxoWellcome),cyclophosphamide (Cytoxan®, Mead Johnson), mecUorethamine (Mustargen®,Merck), melphalan (Alkeran®, GlaxoWellcome), busulfan (Myerlan®,GlaxoWellcome), methotrexate (various), cytarabine (various),fluorouracil (5-fluorouracil) (various, including Adrucil®, PharmaciaUpjohn), cladribine (2-chlorodeoxyadenosine, Leustain®, OrthoBiotech),fludarabine (Fludara®, Berlex) hydroxyurea (Hydrea®, Bristol-MyersSquibb), asparaginase (Elspar®, Berlex), mitoxantrone Novantrone®,Immunex) and procarbazine (Matulane®, Roche). In one aspect, theimmunosuppressive therapy can be an immunosuppressive agent notgenerally used as a antineoplastic or chemotherapeutic agent. Suchimmunosuppressive agents can be alefacept (Amevive®, Biogen),azathioprine (Imuran®, GlaxoWellcome), basiliximab (Simulecf®,Novartis), cyclosporin (Sandimmune® and Neoral®, Sandoz) daclizumab(Zenapax®, Roche), glatiramer (Copaxone®, TEVA), muromonab-CD3(Orthoclone OKT3®, Ortho Biotech), mycophenolate (CellCept®, Roche),tacrolimus (Prograf®, Fujisawa) and sirolimus (Rapamune®, Wyeth Labs).The appropriate immunosuppressive or chemotherapeutic agent can beselected by one of skill in the art based on the individual, forexample, comorbid medical conditions, overall subject health and age.

Other at least one other active agents to be used with a GCSF include ananti-inflammatory agent. The anti-inflammatory agent can be one thatreduces leukocyte populations. Other anti-inflammatory agents can beused as well. For example, vitamin D3 (1,25-dihydroxycholecalciferol)and analogs thereof can be used.

Other at least one other active agents to be used with a GCSF are thosecurrently used to treat recurring spontaneous abortion or miscarriage,preeclampsia, IVIG implantation failure or preterm labor, such asintravenous Ig and heparin.

The invention provides methods of administering compositions of G-CSFuseful for preventing spontaneous abortion or implantation failure ortreating or preventing preeclampsia or preterm labor. In thecompositions administered, the G-CSF can be formulated in any mannerknown to those of skill in the art for formulating and administeringeffective amounts of G-CSF. Preferred formulations include commerciallyavailable formulations.

Filgrastim or G-CSF is available as a preservative pharmaceuticalcomposition comprising 300 mcg/ml. The composition can be administeredsubcutaneously without further admixture. Intravenous preparationsrequire dilution with proper diluent, such as 5% dextrose, diluted to afinal concentration of filgrastim of 5 to 15 mcg/ml. Saline is notrecommended as a diluent due to product precipitation. Mixture withalbumin is recommended to prevent adsorption to plastic materials duringpreparation and infusion. The final concentration of human albuminshould be 2 mg/ml. It is highly recommended that filgrastim berefrigerated at 2° to 8° C.

The presently available pharmaceutical composition contains a smallamount of acetate, Tween 80 and sodium. These excipients are used toachieve and maintain characteristics that are physiologically acceptableto the body and pharmaceutically practical and elegant. Suchcharacteristics include, tonicity, osmoticity, osmolality, osmolarity,viscosity and shelf life. Aqueous pharmaceutical compositions of G-CSFwith increased half life have been described, for example, in U.S. Pat.No. 5,919,757, incorporated herein by reference in its entirety.

The pharmaceutical compositions can comprise the G-CSF in a salt form.For example, because proteins can comprise acidic and/or basic terminiside chains, the proteins can be included in the pharmaceuticalcompositions in either the form of free acids or bases, or in the formof pharmaceutically acceptable salts. Pharmaceutically acceptable saltscan include, suitable acids which are capable of forming salts with theproteins of the present invention including, for example, inorganicacids such as hydrochloric acid, hydrobromic acid, perchloric acid,nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and thelike; and organic acids such as formic acid, acetic acid, propionicacid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonicacid, succinic acid, maleic acid, fumaric acid, cinnamic acid,anthranilic acid, citric acid, naphthalene sulfonic acid, sulfanilicacid and the like. Suitable bases capable of forming salts with thesubject proteins can include, for example, inorganic bases such assodium hydroxide, ammonium hydroxide, potassium hydroxide and the like;and organic bases such as mono-, di- and tri-alkyl amines (for example,triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and thelike) and optionally substituted ethanolamines (for example,ethanolamine, diethanolamine and the like).

Although commercially available G-CSF is currently administeredsubcutaneously or intravenously, any method of administration thatprovides a therapeutically effective amount of G-CSF can be used in themethods of the invention. In one aspect, G-CSF can be in a variety offorms suitable for any route of administration, including, but notlimited to, parenteral, enteral, topical or inhalation.

Parenteral administration refers to any route of administration that isnot through the alimentary canal, including, but not limited to,injectable administration, i.e., intravenous, intramuscular and the likeas described below. Enteral administration refers to any route ofadministration which is oral, including, but not limited to, tablets,capsules, oral solutions, suspensions, sprays and the like, as describedbelow. For purposes of this invention, enteral administration alsorefers to rectal and vaginal routes of administration. Topicaladministration refers to any route of administration through the skin,including, but not limited to, creams, ointments, gels and transdermalpatches, as described below (see also, Pharmaceutical Sciences, 18thEdition (Gennaro et al., eds., Mack Printing Company, Easton, Pa.,1990).

Parenteral pharmaceutical compositions of the present invention can beadministered by injection, for example, into a vein (intravenously), anartery (intraarterially), a muscle (intramuscularly) or under the skin(intradermally or subcutaneously) or in a depot composition.

Injectable pharmaceutical compositions can be sterile suspensions,solutions or emulsions of the G-CSF in aqueous or oily vehicles. Thecompositions can also comprise formulating agents or excipients, such assuspending, stabilizing and/or dispersing agents. The formulations forinjection can be presented in unit dosage form, in ampules or inmultidose containers, and can comprise added preservatives. In certainembodiments, the pharmaceutical compositions contain buffers such ascitrate, acetate, phosphate, tris(hydroxymethyl)amino methane or THAM(tromethamine).

Depot or sustained release pharmaceutical compositions can be used inthe methods of the invention. For example, continuous release of G-CSFcan be achieved by the conjugation of the G-CSF with a water solublepolymer as described in U.S. Pat. No. 5,320,840.

Injectable compositions can be pharmaceutically appropriate compositionsfor any route of injectable administration, including, but not limitedto, intravenous, intrarterial, intracoronary, pericardial, perivascular,intramuscular, subdermal, subcutaneous and intraarticular.

Alternatively, the injectable pharmaceutical composition can be providedin powder form for reconstitution with a suitable vehicle, including butnot limited to sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, the G-CSF can be lyophilized asappropriate. The pharmaceutical compositions can be supplied in unitdosage forms and reconstituted prior to use in vivo.

For prolonged delivery, the pharmaceutical composition can be providedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the pharmaceutical composition can be formulated with suitablepolymeric or hydrophobic materials as an emulsion in an acceptable oilor ion exchange resins, or as sparingly soluble derivatives; as asparingly soluble salt form of the G-CSF, or derivative, mimetic orvariant thereof. The GCSF can be present in an inert matrix or devicefor implantation to achieve prolonged release.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch that slowly releases the active ingredient forpercutaneous absorption can be used. To this end, permeation enhancerscan be used to facilitate penetration of the G-CSF. A particular benefitmay be achieved by incorporating the G-CSF into a transdermal patch.

For oral administration, the pharmaceutical formulations can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., piegelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art (see, Remington'sPharmaceutical Sciences, 18th edition (Gennaro et al., eds.) MackPrinting Company, Pennsylvania, 1990).

Liquid pharmaceutical compositions for oral administration can take theform of, for example, solutions, syrups or suspensions, or they can be adry product for constitution with water or other suitable vehicle beforeuse. Such liquid pharmaceutical compositions can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid).

The pharmaceutical compositions can also comprise buffer salts,flavoring, coloring and sweetening agents as appropriate. Pharmaceuticalcompositions for oral administration can be suitably prepared to providecontrolled release of the G-CSF.

Enteral pharmaceutical compositions can be suitable for buccaladministration, for example, in the form of tablets, troches orlozenges. For rectal and vaginal routes of administration, the G-CSF canbe prepared as solutions (e.g. for retention enemas), suppositories orointments. Enteral pharmaceutical compositions can be suitable foradmixture in feeding mixtures, such as, for mixture with totalparenteral nutrition (TPN) mixtures or for delivery by a feeding tube(see, Dudrick et al., 1998, Surg. Technol. Int. VII:174-184; Mohandas etal., 2003, Natl. Med. J. India 16(1):29-33; Bueno et al., 2003,Gastrointest. Endosc. 57(4):536-40; Shike et al., 1996, Gastrointest.Endosc. 44(5):536-40).

For administration by inhalation, the G-CSF can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator can be formulatedcomprising a powder mix of the compound and a suitable powder base suchas lactose or starch. Inhaled pharmaceutical compositions can be those,for example, described in U.S. Pat. Nos. 5,284,656 and 6,565,841,incorporated herein by reference in their entirety.

The compositions can, if desired, be presented in a pack or dispenserdevice that can comprise one or more unit dosage forms comprising theG-CSF. The pack can, for example, comprise metal or plastic foil, suchas a blister pack. The pack or dispenser device can be accompanied byinstructions for administration.

The pharmaceutical compositions can be for a single, one time use or cancontain antimicrobial excipients, rendering the composition suitable formultiple, extended use with greater shelf stability, for example, amulti-use bottle. In another embodiment, the pharmaceutical compositionof interest can be in unit dose or unit-of-use packages. As known in theart, a unit dose is targeted for a single use. The unit dose form can bein a vial, which can contain a solution or a desiccated form forreconstitution, a pre-filled syringe, a transdermal patch and the like.

As is known to those of skill in the art, a unit-of-use package is aconvenient prescription size, patient ready unit labeled fordistribution by health care providers. The package contains as muchactive ingredient as necessary for a typical treatment regimen.

The pharmaceutical composition can be labeled and have accompanyinglabeling to identify the composition contained therein and otherinformation useful to health care providers and end users. Theinformation can include instructions for use, dose, dosing interval,duration, indication, side effects and other contraindications,warnings, precautions, storage recommendations and the like.

Various embodiments of the pharmaceutical compositions have beendescribed. The descriptions and examples are intended to be illustrativeof the invention and not limiting. Indeed, it will be apparent to thoseof skill in the art that modifications to the pharmaceuticalcompositions can be made to the various embodiments of the inventiondescribed without departing from the spirit of the invention.

The invention provides methods of administering compositions of G-CSFuseful for preventing spontaneous abortion, implantation failure duringassisted reproduction or treating or preventing preeclampsia. The G-CSFand G-CSF compositions can be administered by any route or on anyschedule which provides a therapeutically or prophylactically effectiveamount of G-CSF.

In one aspect the G-CSF or G-CSF, compositions can be administeredparenterally, for example, subcutaneously or intravenously. Theparenteral administration can be in a single bolus or as a continuousinfusion. In one aspect the parenteral administration can be a singleintravenous infusion given over 15-30 minutes. In another aspect theparenteral administration can be a continuous infusion of G-CSF dilutedin 5% dextrose.

The methods provide for administration of G-CSF for a therapeutically orprophylactically effective time. In certain embodiments, the G-CSF isadministered prior to the onset or observation of the disorder orsymptoms accompanying the disorder. In further embodiments, the G-CSF isadministered during the disorder or during the time period that symptomsaccompanying the disorder are observed. In other embodiments, the G-CSFis administered for a time after the disorder had cleared. For example,the G-CSF can be administered about one day, about two days, about threedays, about four days, about one week, about two weeks and up to abouteight weeks, following resolution of the preeclampsia, signs of pretermlabor, threatened abortion or after confirmation of pregnancy duringassisted reproduction.

In another aspect, the present invention provides kits for carrying outthe methods of the invention. For example, the present inventionprovides kits for preventing spontaneous abortion or implantationfailure during assisted reproduction. The kits comprise one or moreeffective doses of G-CSF along with a label or labeling withinstructions on using the G-CSF to prevent spontaneous abortion orimplantation failure during assisted reproduction according to themethods of the invention. In certain embodiments, the kits can comprisecomponents useful for carrying out the methods such as devices fordelivering the G-CSF. In certain embodiments, the kit can comprisecomponents useful for the safe disposal of devices for delivering theG-CSF, e.g., a sharp container for used syringes.

In other embodiments, the present invention provides kits for preventingspontaneous abortion or implantation failure during assistedreproduction by administering mobilized CD34+ peripheral blood stemcells. These kits comprise one or more effective doses of G-CSF alongwith a label or labeling with instructions on using the G-CSF tomobilize CD34+ peripheral blood stem cells according to the methods ofthe invention. The kits can also comprise a label or labeling withinstructions for collecting and/or storing peripheral blood stem cells.In certain embodiments, the kits comprise a myeloablativeimmunosuppressive agent described above. These kits can also comprisecomponents useful for carrying out the methods such as devices fordelivering the G-CSF and components for the safe disposal of thesedevices. The kits can also comprise devices for collecting blood stemcells and devices and formulations for storing blood stem cells.

In further embodiments, the present invention provides kits forpreventing spontaneous abortion or implantation failure during assistedreproduction by administering G-CSF-mobilized peripheral blood stemcells to a subject. The kits comprise a device for administeringperipheral blood stem cells along with instructions for administeringG-CSF-mobilized peripheral blood stem cells to a subject to preventspontaneous abortion or implantation failure during assistedreproduction. In certain embodiments, the present invention provideskits comprising the components of the kits for mobilizing CD34+peripheral blood stem cells and the components of for administeringG-CSF-mobilized peripheral blood stem cells to a subject.

In further embodiments, the present invention provides kits for treatingor preventing preeclampsia or preterm labor. The kits comprise one ormore effective doses of G-CSF along with a label or labeling withinstructions on using the G-CSF to treat or prevent preeclampsia orpreterm labor according to the methods of the invention. In certainembodiments, the kits can comprise components useful for carrying outthe methods such as devices for delivering the G-CSF and components forthe safe disposal of these devices.

In further embodiments, the instant invention provides kits for treatingor preventing preterm labor. The kits comprise one or more effectivedoses of GCSF along with a label or labeling with instructions on usingthe GCSF to treat or prevent preterm labor according to the methods ofthe invention. In certain embodiments, the kits can comprise componentsuseful for carrying out the methods such as devices for delivering theGCSF and components for the safe disposal of the devices.

The invention now will be exemplified in the following non-limitingexamples.

EXAMPLE 1 G-CSF Prevents Embryotoxic Effects of Cells from Women withRecurrent Spontaneous Abortion In Vitro

G-CSF is effective in preventing the death of mouse embryos in an invitro clinical assay for spontaneous abortion. Mouse bioassays havewidely been used to detect embryotoxic effects of sera from subjectshaving reproductive difficulty. (See, Cameo, et al., 1999, Human Reprod.14(4):959-63, Oksenberg and Brautbar 1986, Am. J. Reprod Immunol.Microbiol. 11 (4):118-24, Roussev et al., 1995, Am. Reprod. Immunol.33(2):171-175 and Thomason et al., 1995, Am. J. Reprod. Immunol.34(6):338-41.).

In the in vitro clinical assay, mononuclear leukocytes are isolated fromwomen suffering from recurrent spontaneous abortion. The leukocytes arecultured, and the culture medium is removed from the leukocytes. Thisculture medium is then contacted with murine embryos. Toxic factors inthe culture medium typically kill the murine embryos in this assay.

The mononuclear leukocytes are incubated with G-CSF prior to removal ofthe culture medium. The culture medium is then removed from theleukocytes and contacted with murine embryos. Survival of the murineembryos indicates the reduction of embryotoxic factors in the culturemedium and thereby the effectiveness of G-CSF administration forprevention of spontaneous abortion in this in vitro model.

EXAMPLE 2 G-CSF Prevents Spontaneous Abortion in a Mouse Model In Vitro

G-CSF effectively inhibits a well-known in vivo model for spontaneousabortion.

The murine mating pair CBA×DBA/2 (see e.g., Yabuki et al., 2003, Exp.Anim. 52(2)159-63) results in a spontaneous abortion rate ofapproximately 40%. In this example, female CBA mice are treatedaccording to the methods of the invention. They are treated with G-CSFprior to mating, at the time of mating and immediately after mating. Areduction of the rate of spontaneous abortion in mice treated with G-CSFrelative to control mice indicates that G-CSF effectively preventsspontaneous abortion in this in vivo model.

EXAMPLE 3 G-CSF Prevents Habitual Abortion In Vivo

Thirty-one women with habitual abortion, having more than threeabortions, were recruited in the study (Scarpellini & Sbracia, 2004, Am.J. Repro. 1 mm. 51(6)433-4). The cytogenetic studies,hysterosalpingography, ultrasound, endometrial biopsy, hormonal assays(estradiol, progesterone, prolactin, thyroid hormones, etc.) diabetesworkup and autoantibody tests (ACA, AND, AMA, SMA and anti-lupus Ab)were unremarkable. All of the women failed a previous treatment withIgs, or corticosteroid and aspirin in the former pregnancy. Sixteenwomen were randomly chosen and treated with filgrastim at 100 mg/day SC,which was started the sixth day after ovulation and continued throughthe 35th day after ovulation. The other 15 women received a placebo andprogesterone.

In the group receiving filgrastim, 14 of 16 became pregnant andmaintained pregnancy during the recording period. The karyotype of thefetuses was normal. In the control group, only four pregnanciesoccurred. hCG levels in the treated women were increased by a third overthe levels observed in the control women.

All publications and patent applications in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference in entirety. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretodeparting from the spirit or scope of the invention described herein.

1. A method of treating or preventing preeclampsia in a subject in needthereof comprising administering to the subject an effective amount ofG-CSF.
 2. The method of claim 2, wherein the subject is in the second orthird trimester of pregnancy.
 3. The method of claim 1, wherein theG-CSF is administered parenterally.
 4. The method of claim 1, whereinthe G-CSF is administered subcutaneously.
 5. A kit for treating orpreventing preeclampsia comprising an effective amount of G-CSF and alabel or labeling with instructions for using the G-CSF to treat orprevent preeclampsia.
 6. A method of treating or preventing pretermlabor in a subject in need thereof comprising administering to thesubject an effective amount of G-CSF.
 7. The method of claim 6, whereinthe G-CSF is administered parenterally.
 8. The method of claim 6,wherein the G-CSF is administered subcutaneously.
 9. A kit for treatingor preventing preterm labor comprising an effective amount of G-CSF anda label or labeling with instructions for using the G-CSF to treat orprevent preterm labor.
 10. A method for identifying a patient at risk ofhaving difficulty in sustaining a pregnancy, having difficulty in havingin vitro fertilized embryos implanted, having preeclampsia or havingpremature labor comprising: determining, in a tissue sample from saidpatient, at least one of the parameters selected from the groupconsisting of the Th1 cell number, Th2 cell number, Th1 cell activity,Th2 cell activity, the amount of a Th1 cell product, and the amount of aTh2 cell product; and identifying the patient as one having a high riskof having difficulty in sustaining a pregnancy, having difficulty inhaving in vitro fertilized embryos implanted, having preeclampsia orhaving premature labor if the patient meets one or more of the criteriaselected from the group consisting of elevated Th1 cell number, elevatedTh1 cell activity, elevated amount of a Th1 cell product, decreased Th2cell number, decreased Th2 cell activity, decreased amount of a Th2 cellproduct, a higher ratio of Th1 cell number to Th2 cell number, and ahigher ratio of Th1 cell activity.
 11. The method of claim 10, whereinthe Th1 cell product is interferon γ.
 12. The method of claim 10,wherein the Th2 cell product is interleukin (IL)-10.
 13. The method ofclaim 1, wherein the G-CSF is administered in combination with animmunosuppressive agent.
 14. The method of claim 6, wherein the G-CSF isadministered in combination with an immunosuppressive agent.
 15. Themethod of claim 10, wherein the Th1 cell product is tumor necrosisfactor β.
 16. The method of claim 10, wherein the Th1 cell product isIL-2.
 17. The method of claim 10, wherein the Th2 cell product isselected from the group consisting of IL-4, IL-5, IL-6, IL-10, IL-13,and combinations thereof.
 18. The method of claim 10, wherein the Th2cell product is IL-4, IL-10 or combinations thereof.
 19. The method ofclaim 10, wherein the Th2 cell product is IL-4.
 20. The method of claim10, wherein said tissue sample is a blood sample.